Expression Of Equilibrative Nucleoside Transporter Research Articles

Efficacy of Low-Dose Oral Metronomic Dosing of the Prodrug of Gemcitabine, LY2334737, in Human Tumor Xenografts

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.

3′-Deoxy-3′-[18F]-fluorothymidine ([18F]-FLT) transport in newly diagnosed glioma: correlation with nucleoside transporter expression, vascularization, and blood–brain barrier permeability

3'-Deoxy-3'-[(18)F]-fluorothymidine ([(18)F]-FLT), a marker of cellular proliferation, has been used in positron emission tomography (PET) examination of gliomas. The aim of this study was to investigate whether the uptake of [(18)F]-FLT in glioma correlates with messenger RNA (mRNA) levels of the equilibrative nucleoside transporter 1 (ENT1), microvascular density (assessed by CD34 immunohistochemistry), and the blood-brain barrier (BBB) breakdown. A total of 21 patients with newly diagnosed glioma were examined with [(18)F]-FLT PET. Tumor lesions were identified as areas of focally increased [(18)F]-FLT uptake, exceeding that of surrounding normal tissue. Dynamic analysis of [(18)F]-FLT PET revealed correlations between the phosphorylation rate constant k 3 and ENT1 expression; however there was no correlation between the kinetic parameters and CD34 score. There was a good correlation between the gadolinium (Gd) enhancement score (evaluating BBB breakdown) and ENT1 expression, CD34 score, and Ki-67 index. This preliminary study suggests that ENT1 expression might not reflect accumulation of [(18)F]-FLT in vivo due to BBB permeability in glioma.

Expression of equilibrative nucleoside transporter 1 in rat circumvallate papillae

In gustatory function, communication between four types taste buds cells plays crucial roles. ATP is one of the intercellular signaling molecules in taste buds, and the extracellular ATP fate is regulated by its cellular clearance, but there is little information on it. Therefore, we examined the expression profiles of nucleoside transporters (NTs) as a clearance system for ATP metabolite adenosine in rat circumvallate papillae (CP) by RT-PCR, real-time PCR and immunohistochemistry. Among NTs, mRNA for Ent1 was expressed by the CP, and significantly was greater in the CP as compared with non-CP. ENT1 immunoreactivity was detected in PLC-β2-positive type II (71.0±8.5%), chromogranin-A-positive type III (64.9±7.4%), and SNAP25-positive type III (77.0±10.4%) taste cells, but not in NTPDase2-positive type I ones. These results indicate that ENT1-expressing type II and III taste cells might comprise an adenosine clearance system in taste buds of the CP. ENT1 expression in taste cells is important for elucidation of complicated taste signaling.

P63 Ribavirin uptake by primary human hepatocytes corresponds to equilibrative nucleoside transporter 1 expression

IntroductionCombined therapy with pegylated interferon (INF) and ribavirin (RV) is the current standard of care for HCV infection, with a 40% to 80% success rate depending on viral genotype. The...

In-Vitro Efficacy of the Deoxyguanoside Analogs Forodesine (BCX-1777) and ARA-G in Pediatric Acute Lymphoblastic Leukemia.

Abstract 2038Poster Board II-15Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with elevated deoxyguanosine (dGuo) plasma levels. DGuo is converted into dGTP inducing apoptosis in T-cells and this provides the rationale for the development of deoxyguanosine analogues as a potential treatment option for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity, thereby boosting the conversion of dGuo into dGTP and raising intracellular dGTP levels. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation that is efficiently converted into AraGTP. AraGTP becomes incorporated in the DNA, blocking DNA synthesis and promoting apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of Forodesine treatment in pediatric ALL patients are not yet available.As tested on primary pediatric acute lymphoblastic leukemia (ALL) patient samples (4 T-ALL, 2 BCP-ALL), 1μM of Forodesine is sufficient to completely block PNP and abolish rapid dGuo degradation resulting in a median 7.9 (range 0.5-378) fold raise of intracellular dGTP levels. Accumulation of dGTP is comparable for T-ALL (n=31) and BCP-ALL (n=11) patient samples. This reflects equal intrinsic ability of salvage nucleotide synthesis for both T-ALL and BCP-ALL cells. Cytotoxic effect of Forodesine was tested on primary leukemia cells from newly diagnosed pediatric ALL patients in-vitro by incubating cells with Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001-50μM). In accordance with selective T-cell toxicity, T-ALL cells were more sensitive to Forodesine/dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to BCP-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5-222.1 fold), although the raise of dGTP levels did not correlate with Forodesine/dGuo toxicity (r2= 0.10, p=0.22).Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to BCP-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for BCP-ALL samples (n=20). Remarkably, TELAML1 positive BCP-ALL cases were insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities (r2=0.05, p=0.29). Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment. This may be explained by the fact that the uptake of both drugs may be facilitated by different transporters. Using RQ-PCR we could demonstrate that AraG toxicity, in contrast to Forodesine, was significantly associated with ENT1 (equilibrative nucleoside transporter 1) expression levels (p=0.008), which was previously identified as strong predictor for AraC cytotoxicity in pediatric ALL (Stam RW. et al., Blood 2003). AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p<0.0001). We found no significant correlation between Forodesine sensitivity and the expression levels of other nucleoside transporters (CNT1, CNT2, CNT3, ENT2), kinases (dCK, dGK), nucleotidases (NT5C1A, NT5C2, PNI) or other enzymes that are involved in dGuo metabolism (PNP, RRM1, RRM2).In conclusion, T-ALL cells are more sensitive to Forodesine/dGuo treatment in-vitro than BCP-ALL cells that have nearly 8 fold higher dGuo LC50 values. Resistance to AraG treatment does not preclude responsiveness to Forodesine treatment and vice versa, indicating that Forodesine and AraG rely on different cellular mechanisms for cytotoxicity, possibly involving differences in dependence on the nucleoside transporter ENT1. Disclosures:No relevant conflicts of interest to declare.

FLT3-ITD induces ara-C resistance in myeloid leukemic cells through the repression of the ENT1 expression

Fms-related tyrosine kinase 3-internal tandem duplications ( FLT3-ITD) are strongly associated with the refractory nature of acute myeloid leukemia (AML) by the standard combined chemotherapy. FLT3-ITD-expressing murine and human myeloid cell lines, HF6/FLT3-ITD and K562/FLT3-ITD cells, respectively, were developed in order to clarify whether FLT3-ITD is involved in the resistance to cytotoxic agents in AML. Both of these cell lines were specifically resistant to the pyrimidine analogue cytosine arabinoside (ara-C), an essential agent for AML, accompanied by the downregulation of equilibrative nucleoside transporter 1 (ENT1), a transporter responsible for the cellular uptake of ara-C. The ENT1 promoter activity and the cellular uptake of ara-C were reduced in K562/FLT3-ITD cells, and rescued by pretreating the cells with PKC412, a FLT3 inhibitor. In addition, the expression of hypoxia inducible factor 1 alpha subunit ( HIF1A) transcripts was upregulated in K562/FLT3-ITD cells, and the induction of HIF-1α reduced the promoter activity of ENT1 gene in K562 cells. Taken together, these findings suggest that FLT3-ITD specifically induces ara-C resistance in leukemic cells by the repression of ENT1 expression, possibly through the upregulation of HIF-1α, while also partially accounting for the poor prognosis of AML with FLT3-ITD due to resistance to the standard chemotherapy protocols which include ara-C.

Equilibrative Nucleoside Transporters in Fetal Endothelial Dysfunction in Diabetes Mellitus and Hyperglycaemia

Diabetes mellitus types 1 and 2, and gestational diabetes are characterized by abnormal D-glucose metabolism and hyperglycaemia, and induce foetal endothelial dysfunction with implications in adult life increasing the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (i.e. the L-arginine/NO signalling pathway) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pathological pregnancies, including pregnancies with pre-established diabetes mellitus or in gestational diabetes. The mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), amino acid transporters and NO synthases (NOS). Modulation of ENTs and NOS expression and activity in endothelium involves several signalling molecules, including protein kinase C, mitogen-activated protein kinases p42 and p44, calcium and phosphatidyl inositol 3 kinase. Elevated extracellular D-glucose and diabetes alters human endothelial function. However, information regarding modulation the transport capacity as well as expression of ENTs is limited. This review focuses on the effect of diabetes mellitus and gestational diabetes, and hyperglycaemia on the reported mechanisms described for transcriptional and post-transcriptional regulation of ENTs, and the potential consequences for foetal endothelial function in these pathologies. Recent available information regarding functional consequences of an abnormal environment on the functionality of the endothelium from microvasculature of the human placenta is mentioned. The available information is scarce, but it could contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in this pathological conditions, emphasizing the key role of this type of epithelium in fetal-placental function and the normal foetal development and growth.

Pharmacogenetics in oncology

The number of studies reporting a relationship between DNA sequence variants and cancer treatment outcome is increasing. In particular, the following associations were found: dihydropyrimidine dehydrogenase (DPD) gene mutations and severe 5-FU toxicity, epidermal growth factor receptor mutations (EGFR) and responsiveness of NSCLC to gefitinib, ERCC1 polymorphisms and activity of cisplatin, genetic variants of UGT1A1 gene and severe neutropenia by irinotecan, thymidylate synthase (TS) gene polymorphisms and 5-FU sensitivity, and cytidine deaminase (CDA) genotype and expression of equilibrative nucleoside transporter-1 (hENT1) and response to gemcitabine. The next step in pharmacogenetic research should be the validation of these findings in randomised prospective trials, specifically designed to compare the outcome of treatment selected on the basis of patient’s genotype versus standard approach. In conclusion, the improvement in genotyping technologies, combined with efficient and cost-effective analytical methods, may fulfil the promise of personalising the treatment offered to cancer patients.

Amino acid residue 24 of the human equilibrative nucleoside transporter 1 (ENT1) is important for transport activity

Nucleoside analogs such as cytarabine (Ara‐C) and gemcitabine are commonly used to treat cancer (leukemia and breast cancer among others). It has been previously shown that a correlation between drug resistance and reduced uptake of these agents is due to decreased expression of a major equilibrative nucleoside transporter, ENT1. [3H]‐Uridine uptake and [3H]‐NBMPR binding revealed that the CEM‐AraC/8C cells, a leukemia cell line resistant to Ara‐C, are devoid of functional ENTs. Sequence analysis for ENT1 gene from CEM‐AraC/8C revealed a missense point mutation in the ENT1 gene sequence that confers a glycine to arginine substitution at residue 24. To determine the importance of G24 to ENT1 transport activity G24R, G24E, and G24A ENT1 mutants were expressed in PK15 cells, an ENT‐deficient cell line. Expression of wt ENT1 produced a time‐dependent increase in [3H]‐uridine uptake whereas cells that expressed G24R ENT1 were devoid of [3H]‐uridine uptake. Expression of G24E and G24A ENT1 led to only partial [3H]‐uridine incorporation, compared to wt ENT1. Furthermore, the expression of wt ENT1 in PK15 cells increased nucleoside analog‐induced cell death whereas a reduced effect was observed during the expression of the ENT1 mutants. This study reveals that CEM‐AraC/8C resistance to Ara‐C may be attributed to the G24R mutation in ENT1 and that this single amino acid is important for ENT1 nucleoside transport activity.

High glucose suppresses expression of equilibrative nucleoside transporter 1 (ENT1) in rat cardiac fibroblasts through a mechanism dependent on PKC‐ζ and MAP kinases

Recently it was demonstrated that the elevated concentration of glucose but not lack of insulin is responsible for suppression of equilibrative nucleoside transporter (ENT1) in diabetic rat cardiac fibroblasts (CFs). The present study was undertaken to determine the signaling pathway utilized by glucose to regulate the expression of ENT1 in the primary culture of rat CFs. Pretreatment of CFs with Go 6983, an isozyme non-selective PKC inhibitor, prevented the high glucose (25 mM) effect on ENT1 mRNA level and nitrobenzylthioinosine (NBTI)-sensitive adenosine uptake. Similar effect was observed with a cell-permeable PKC-zeta pseudosubstrate, whereas Go 6976 a selective inhibitor of Ca(2+)-dependent PKC-alpha and PKC-beta isozymes had little effect on high glucose-induced suppression of ENT1 mRNA level. Incubation of CFs with nitric oxide (NO) donors (SNAPE, SNP) or NO synthase inhibitors (L-NAME, L-NMMA) prior to exposition of CFs to high glucose did not change the glucose effect on ENT1 mRNA level. The high glucose-induced suppression of ENT1 expression was blocked by PD9859 (an inhibitor of MEK), whereas neither wortmannin (an inhibitor of PI3K) nor rapamycin (an inhibitor of mTOR) affected the glucose action on ENT1 transcript level. Highly effective in preventing the high glucose effect on ENT1 mRNA level were GW 5074 (an inhibitor of Raf kinase) and SB 203580 (selective p38 MAPK inhibitor). These findings indicate that high glucose suppresses the expression of ENT1 in CFs by NO independent manner involving the signaling through PKC-zeta, Raf-1, MEK, and p38 MAPK pathways.

Cellular localization and functional characterization of the equilibrative nucleoside transporters of antitumor nucleosides

Nucleoside transporters play an important role in the disposition of nucleosides and their analogs. To elucidate the relationship between chemosensitivity to antitumor nucleosides and the functional expression of equilibrative nucleoside transporters (ENT), we established stable cell lines of human fibrosarcoma HT-1080 and gastric carcinoma TMK-1 that constitutively overexpressed green fluorescent protein-tagged hENT1, hENT2, hENT3 and hENT4. Both hENT1 and hENT2 were predictably localized to the plasma membrane, whereas hENT3 and hENT4 were localized to the intracellular organelles. The chemosensitivity of TMK-1 cells expressing hENT1 and hENT2 to cytarabine and 1-(3-C-ethynyl-beta-D-ribopentofuranosyl) cytosine increased markedly in comparison to that of mock cells. However, no remarkable changes in sensitivity to antitumor nucleosides were observed in cell lines that expressed both hENT3 and hENT4. These data suggest that hENT3 and hENT4, which are mainly located in the intracellular organelles, are not prominent nucleoside transporters like hENT1 and hENT2, which are responsible for antitumor nucleoside uptake.

Equilibrative Nucleoside (ENTs) and Cationic Amino Acid (CATs) Transporters: Implications in Foetal Endothelial Dysfunction in Human Pregnancy Diseases

Gestational diabetes (GD, characterized by abnormal D-glucose metabolism), intrauterine growth restriction (IUGR, a disease associated with reduced oxygen delivery (hypoxia) to the foetus), and preeclampsia (PE, a pregnancy complication characterized by high blood pressure, proteinuria and increased vascular resistance), induce foetal endothelial dysfunction with implications in adult life and increase the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (the NO synthase (NOS) substrate) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pregnancies with GD, IUGR or PE. Mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), cationic amino acid transporters (CATs), and NOS. Modulation of ENTs, CATs, and NOS expression and activity in endothelium involves protein kinase C (PKC), mitogen-activated protein kinases p42 and p44 (p42/44(mapk)), calcium, and phosphatidyl inositol 3 kinase (PI3k), among others. Elevated extracellular D-glucose and hypoxia alter human endothelial function. However, information regarding the transcriptional modulation of ENTs, CATs, and NOS is limited. This review focuses on the effect of transcriptional and post-transcriptional regulatory mechanisms involved in the modulation of ENTs and CATs, and NOS expression and activity, and the consequences for foetal endothelial function in GD, IUGR and PE. The available information will contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in these pregnancy diseases and will emphasize the key role of this type of epithelium in placental function and the normal foetal development and growth.

Schedule-dependent antitumor effects of gemcitabine and S-1, a novel oral derivative of 5-fluorouracil, in human pancreatic cancer xenografts

4154 Background: The systemic administration of gemcitabine (GEM) has been accepted as a standard treatment for patients with advanced pancreatic cancer. The major mediator of cellular uptake of GEM is the equilibrative nucleoside transporter 1 (ENT1) which expression is up-regulated by TS inhibitor such as 5-fluorouracil (5-FU). S-1 is a novel oral derivative of 5-FU prodrug tegafur combined with two modulators, 5-chloro-2, 4-dihydropridine and potassium oxonate, and recent clinical trials have reported the promising effect of S-1 in pancreatic cancer. The purpose of this study is to evaluate the relationship between different schedules and their effects of GEM/S-1 in combination therapy for pancreatic cancer in nude mouse xenograft model. Methods: Expression of ENT1 was determined by quantitative RT-PCR. GEM cellular uptake was determined using [3H]GEM. Seven pancreatic cancer cell lines (AsPC1, BxPC3, MiaPaCa-2, PSN1, Panc1, PCI6, and KMP4) were treated in vitro with 5-FU either before or following exposure to GEM. Growth inhibitory effects in vitro were determined by MTT assay. We compared 6 different treatment schedules (no treatment, single agent of GEM or S-1, combination treatment with gemcitabine either before, simultaneously or following administration of S-1) using MiaPaCa-2 xenograft model in BALB/c mice. The antitumor effects were evaluated with the tumor volume. Results: Significant increases in ENT1 expression and GEM cellular uptake were observed after 5-FU treatment in vitro and S-1 treatment in vivo. The in vitro growth inhibitory effect was significantly greater in the sequential treatment of 5-FU followed by GEM in all cell lines except for Panc1. Furthermore, the significant tumor growth inhibition in vivo was observed in the mice treated with S-1 followed by GEM compared with either untreated mice or the mice treated with gemcitabine followed by S-1. Conclusions: Based on the modulation of 5-FU on the uptake of GEM, 5-FU should be used before GEM treatment. The combination therapy with preadministarted S-1 and GEM in the patients with pancreatic cancer is promising and is worthwhile to be confirmed in clinical trials. No significant financial relationships to disclose.

D-Glucose upregulates adenosine transport in cultured human aortic smooth muscle cells

The etiology of the atherosclerosis that occurs in diabetes mellitus is unclear. Adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM d-glucose, which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT-1) and ENT-2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT-1 and inhibited by nitrobenzylmercaptopurine riboside (NBMPR, IC(50) = 0.69 +/- 0.05 nM). Adenosine transport in HASMCs was increased by >30% after treatment for 48 h with 25 mM d-glucose, but not with equimolar d-mannitol and l-glucose. Kinetic studies showed that d-glucose increased V(max) of adenosine transport without affecting K(m). Similarly, d-glucose increased B(max) of high-affinity [(3)H]NBMPR binding, while the dissociation constant (K(d)) was not changed. Consistent with these observations, 25 mM d-glucose increased mRNA and protein expression of ENT-1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD-98059 (40 microM) and U-0126 (10 microM), abolished the effect of d-glucose on ENT-1. We conclude that d-glucose upregulates the protein and message expression and functional activity of ENT-1 in HASMCs, possibly via MAPK/ERK-dependent pathways. Pathologically, the increase in ENT-1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and, thus, alter vascular functions in diabetes.